US20140243319A1 - Treatments targeting gamma-synuclein expression levels - Google Patents

Treatments targeting gamma-synuclein expression levels Download PDF

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US20140243319A1
US20140243319A1 US14/005,753 US201214005753A US2014243319A1 US 20140243319 A1 US20140243319 A1 US 20140243319A1 US 201214005753 A US201214005753 A US 201214005753A US 2014243319 A1 US2014243319 A1 US 2014243319A1
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Anita Sidhu
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • compositions that modulate expression of ⁇ -synuclein and methods of treatment comprising modulation of expression of ⁇ -synuclein are described.
  • TCA tri-cyclic antidepressants
  • a compound that reduces ⁇ -synuclein expression in combination with chemotherapeutic compounds can provide a synergistic cell killing effect that is greater than the effect provided by administering either compound by itself.
  • a combination treatment taking advantage of the synergistic effect of TCA's and chemotherapeutic agents to provide improved treatment of cancers, preferably stage III/IV cancers, can include determining that the cancer cells to be treated are expressing ⁇ -Syn. Determination of ⁇ -Syn expression can be by direct assay of a cell sample. For example, the presence of ⁇ -Syn can be determined by Western blot, another obseravtion of antibody binding, or other biochemical assay. Alternatively, determination of ⁇ -Syn expression can be accomplished by determining the presence in the cells of RNA encoding ⁇ -Syn.
  • Determination of ⁇ -Syn can also be accomplished by observation of characteristics associated with ⁇ -Syn expressing cancer cells, for example cell morphology, cytometric observations, staining, phenotypes associated with ⁇ -Syn expression, resistance to treatment with taxanes.
  • a combination treatment taking advantage of the synergistic effect of TCA's and chemotherapeutic agents to provide improved treatment of cancers can include co-administering, to a patient in need thereof, an effective amount of a chemotherapeutic agent, preferably a MT targeting agent, and most preferably a taxane or taxane derivative together with an effective amount of a ⁇ -Syn expression inhibitor, preferably a TCA or derivative thereof such as DMI.
  • the combination treatment can include administering a ⁇ -Syn expression inhibitor, preferably a TCA or derivative thereof, such as DMI prior to administration of the chemotherapeutic agent, such that ⁇ -Syn expression is inhibited at the time that the chemotherapeutic agent is administered.
  • a ⁇ -Syn expression inhibitor preferably a TCA or derivative thereof, such as DMI
  • the administration of—Syn expression inhibitor and chemotherapeutic agents can be repeated in alternating or overlapping cycles to achieve a further effect.
  • a method for identifying improved methods of killing cancer cells that express ⁇ -Syn can comprise determining the ability of an agent to inhibit ⁇ -Syn expression in a ⁇ -Syn expressing cancer cell and determining the ability of the ⁇ -Syn expression inhibiting agent to enhance the cytotoxic effectiveness of a chemotherapeutic agent such as a taxane or taxane derivative in killing ⁇ -Syn expressing cancer cells.
  • An improved method of killing cancer cells can comprise co-administration of an agent effective to inhibit ⁇ -Syn expression identified by such a method with a chemotherapeutic agent whose effectiveness is increased by inhibition of ⁇ -Syn, or pretreatment with the agent effective to inhibit ⁇ -Syn expression followed by administration or co-administration of an effective amount of the chemotherapeutic agent such that ⁇ -Syn expression is inhibited at the time that the chemotherapeutic agent is administered.
  • FIG. 1 shows improved behavior in WKY rats treated with antidepressants.
  • FIG. 2 shows that antidepressants reduce ⁇ -Syn levels in the brain of WKY rats.
  • FIG. 3 shows—Syn protein levels are reduced in brain of WKY rats after desipramine treatment.
  • FIG. 4 shows Western blot data demonstrating ⁇ -Syn expression in various cancer cell lines.
  • FIG. 5 shows that T47D breast cancer cells expressing ⁇ -Syn are resistant to taxol.
  • FIG. 6 shows that desipramine reduces ⁇ -Syn levels in T47D breast cancer cells.
  • FIG. 7 shows results of a viability assay of T47D cells treated with desipramine and taxol.
  • FIG. 8 shows results of a viability assay of SK-BR-3 cells treated with desipramine and taxol.
  • FIG. 9 shows results of a viability assay of HT-29 cells treated with desipramine and taxol.
  • FIG. 10 shows results of a viability assay of HCT-116 cells treated with desipramine and taxol.
  • FIG. 11 shows results of a viability assay of A549 cells treated with desipramine and taxol.
  • FIG. 12 shows results of a viability assay of T47D cells treated with imipramine and taxol.
  • FIG. 13 shows Western blot data showing expression of ⁇ -Syn pancreatic cancer cells
  • FIG. 14 shows results of a viability assay of COLO-357 cells treated with desipramine and taxol.
  • FIG. 15 shows results of a viability assay of BXPC-3 cells treated with desipramine and taxol.
  • FIG. 16 shows results of a viability assay of ASPC-1 cells treated with desipramine and taxol.
  • the synucleins are a family of small soluble proteins that are normally expressed in presynaptic neurons in the brain.
  • ⁇ -synuclein ⁇ -Syn
  • ⁇ -Syn has been linked to the genesis of neurodegenerative disorders, such as Parkinson's disease.
  • ⁇ -synuclein ⁇ -Syn is overexpressed in the brain.
  • DMI desipramine
  • ⁇ -Syn is also known as breast carcinoma specific gene. It was initially isolated from infiltrating breast carcinoma cells. ⁇ -Syn is not normally expressed in normal breast tissue, benign tumors or stage I/II cancers, and its expression in breast cancer (BC) is strongly associated with advanced stages of disease progression, where it has been found to be overexpressed in >70% of Stage III/IV breast and ovarian tumors. ⁇ -Syn overexpression is also seen in a wide variety of other carcinomas such as colorectal, bladder, pancreatic, glaucoma, brain tumor and prostate cancer, where disease progression to stage III/IV is correlated with overexpression of this protein.
  • ⁇ -Syn In breast carcinoma and ovarian carcinoma, the aberrant expression of ⁇ -Syn is thought to be promoted by hypomethylation of the CpG islands of the gamma-synuclein gene. Once overexpressed, gamma-synuclein is thought to promote cancer cell survival and inhibit stress- and chemotherapy drug-induced apoptosis by modulating MAPK pathways.
  • ⁇ -Syn is an oncogene that is overexpressed in >70% of stage III/IV carcinomas but not in stage I or II cancers.
  • Some of the cancers to which the overexpression of ⁇ -Syn is linked to include breast, ovarian, colorectal, bladder, pancreatic, glaucoma, brain tumor and prostate cancer.
  • Patients with ⁇ -Syn positive cancers have a significantly shorter disease-free survival and overall survival, compared to patients that do not express ⁇ -Syn.
  • Microtubule (MT) targeting agents such as taxanes
  • MT targeting agents are currently the first line of chemotherapeutic agents to treat patients with advanced or metastatic cancer, but stage III/IV patient response to taxanes varies significantly.
  • These MT targeting agents rely heavily on the normal function of the mitotic checkpoint machinery, including BubR1, a mitotic checkpoint kinase whose activity is inhibited by ⁇ -Syn.
  • BubR1 a mitotic checkpoint kinase whose activity is inhibited by ⁇ -Syn.
  • the interaction of ⁇ -Syn with BubR1 results in impairment of the mitotic checkpoint machinery, rendering these cells resistant to MT destabilizers.
  • ⁇ -Syn results in increased resistance of these cells to MT targeting agents.
  • in vitro studies pretreating cells with either the cytokine oncostatin M or injecting cells individually with a small anti- ⁇ -Syn peptide have shown that inhibition of ⁇ -Syn expression increases cell susceptibility to MT destabilizing agents.
  • none of these methods of decreasing ⁇ -Syn levels are suitable for treatment of patients.
  • ⁇ -Syn promotes cell survival and proliferation, inhibits stress and current MT inhibitory drug-induced apoptosis, activates estrogen receptor, inhibits mitotic checkpoint control and promotes metastasis in a nude mouse model.
  • Stage III/IV cancers are more resistant to current MT targeting chemotherapies, such as taxol, and in breast cancer cells overexpressing ⁇ -Syn, the presence of ⁇ -Syn renders these cells resistant to these agents.
  • decreasing the expression of ⁇ -Syn in cells through an anti- ⁇ -Syn peptide injected into cells, or upon pretreatment of cells with the cytokine oncostatin M, renders these cells much more susceptible to taxol.
  • treatment strategies that reduce ⁇ -Syn expression levels can provide improved treatment of stage III/IV cancers.
  • Pancreatic ductal adenocarcinoma is currently the fourth leading cause of cancer-related death in Western countries. Long-term survival is rare, with the overall 5-year survival rates ranging from 10% to 25%. In normal pancreas, islet cells and some acinar cells express low levels of ⁇ -Syn but the ductal epithelium is negative for ⁇ -Syn. Very little is known about the role of ⁇ -Syn in PC. In one study, 22 of 32 pancreatic tumor tissue samples (69%) were found to express ⁇ -Syn.
  • ⁇ -Syn was shown to be present in 61% of the tumor tissue samples examined from patients with Stage I and II pancreatic carcinoma.
  • the overexpression of ⁇ -Syn was correlated with perineural and lymph node invasion. This finding was subsequently also confirmed by another study on ⁇ -Syn in pancreatic cancer.
  • proteomics and transcriptomics ⁇ -Syn was found to be the only protein that was up-regulated in high perineural invasive PC, confirming a role for this protein in pancreatic carcinoma invasion.
  • ⁇ -Syn was detected in serum samples from 21 of 56 patients (38%) with pancreatic carcinoma, suggesting that this protein can be a useful biomarker.
  • DMI an FDA-approved tricyclic antidepressant
  • ⁇ -Syn can reduce ⁇ -Syn expression in PC cells at clinically relevant concentrations and cause cell death (>60%). Even low concentrations of the antidepressant, when given in combination with taxol, results in >60-80% cell death of PC cells.
  • overexpression of ⁇ -Syn is also present in a large variety of other stage III/IV cancers, for example, breast, ovarian, colorectal, bladder, pancreatic, glaucoma, brain tumor and prostate cancer. Consequently, DMI treatment, or DMI pretreatment, followed by taxol, can provide improvements in the treatment of other advanced stage III/IV metastatic cancers as well. Since the treatment can use FDA-approved drugs, and is used at clinically relevant doses, this treatment can be immediately used. More generally, these findings demonstrate that treatment of advanced stage III/IV metastatic cancers.
  • ⁇ -Syn was found to be the only protein overexpressed in high perineural invasive PCs, an especially aggressive form of this cancer. Multivariate analyses revealed ⁇ -Syn overexpression as the only independent predictor of diminished overall survival and the strongest negative indicator of disease-free survival associated with PC.
  • ⁇ -Syn is expressed in presynaptic terminals of monoaminergic neurons, and our lab has shown that this protein can regulate the function of norepinephrine and serotonin transporters. Unlike ⁇ -Syn, whose role in neurodegenerative diseases such as Parkinson's disease is well established, a role for ⁇ -Syn in neurodegeneration is not known. ⁇ -Syn is overexpressed in the frontal cortex of the WKY, a rat model of depression. Chronic treatment of the WKY rat with DMI for 2 weeks, caused a decrease in ⁇ -Syn levels in frontal cortex of brain.
  • DMI norepinephrine transporter
  • NET norepinephrine transporter
  • ⁇ -Syn to bind tightly to MTs may be one mechanism by which its overexpression in carcinomas protects cells from the MT actions of chemotherapeutic agents, for example, taxol, nocodazole, vinblastine and colchicine.
  • DMI is a tricyclic antidepressant.
  • Tricyclic antidepressants are a class of psychoactive drugs used primarily as antidepressants. They are named after their chemical structure, which contains three rings of atoms.
  • TCAs include the following: tertiary amines, e.g., Amitriptyline (Elavil), Amitriptylinoxide (Ambivalon, Equilibrin), Butriptyline (Evadyne), Clomipramine (Anafranil); Dosulepin/Dothiepin (Prothiaden), Doxepin (Adapin, Sinequan), Imipramine (Tofranil), Imipraminoxide (Imiprex, Elepsin), Lofepramine (Lomont, Gamanil), Trimipramine (Surmontil), and secondary amines, e.g., Desipramine (Norpramin, Pertofrane); Nortriptyline (Pamelor, Aventyl); Protriptyline (Vivactil); and also includes: Demexiptiline (Deparon, Tinoran), Dibenzepin (Noveril, Victoril), Dimetacrine (Istonil, Iston
  • TCAs are generally understood to act primarily as serotonin-norepinephrine reuptake inhibitors (SNRIs) by blocking the serotonin transporter (SERT) and the norepinephrine transporter (NET), respectively, which results in an elevation of the extracellular concentrations of these neurotransmitters, and therefore an enhancement of neurotransmission.
  • SNRIs serotonin-norepinephrine reuptake inhibitors
  • SERT serotonin transporter
  • NET norepinephrine transporter
  • TCAs also have high affinity as antagonists at the 5-HT2 (5-HT2A and 5-HT2C), 5-HT6, 5-HT7, al-adrenergic, and NMDA receptors, and as agonists at the sigma receptors ( ⁇ 1 and ⁇ 2), some of which may contribute to their therapeutic efficacy, as well as their side effects.
  • the TCAs also have varying but typically high affinity for antagonizing the H1 and H2 histamine receptors, as well as the muscarinic acetylcholine receptors. As a result, they also act as potent antihistamines and anticholinergics. Most, if not all, of the TCAs also potently inhibit sodium channels and L-type calcium channels, and therefore act as sodium channel blockers and calcium channel blockers, respectively.
  • chemotherapeutic agents for use in combination with TCA's are the MT targeting compounds, for example taxanes and derivatives thereof.
  • the taxanes are diterpenes produced by the plants of the genus Taxus (yews). As their name suggests, they were first derived from natural sources, but some have been synthesized artificially. Taxanes include Docetaxel, Larotaxel, Ortataxel, Paclitaxel (Taxol), Tesetaxel, and Epothilones (Ixabepilone).
  • a combination treatment taking advantage of the synergistic effect of TCA's and chemotherapeutic agents to provide improved treatment of cancers, preferably stage III/IV cancers, can include determining that the cancer cells to be treated are expressing ⁇ -Syn. Determination of ⁇ -Syn expression can be by direct assay of a cell sample. For example, the presence of ⁇ -Syn can be determined by Western blot, another obseravtion of antibody binding, or other biochemical assay. Alternatively, determination of ⁇ -Syn expression can be accomplished by determining the presence in the cells of RNA encoding ⁇ -Syn.
  • Determination of ⁇ -Syn can also be accomplished by observation of characteristics associated with ⁇ -Syn expressing cancer cells, for example cell morphology, cytometric observations, staining, phenotypes associated with ⁇ -Syn expression, resistance to treatment with taxanes.
  • a combination treatment taking advantage of the synergistic effect of TCA's and chemotherapeutic agents to provide improved treatment of cancers can include co-administering, to a patient in need thereof, an effective amount of a chemotherapeutic agent, preferably a MT targeting agent, and most preferably a taxane or taxane derivative together with a ⁇ -Syn expression inhibitor, preferably a TCA or derivative thereof such as DMI.
  • a chemotherapeutic agent preferably a MT targeting agent, and most preferably a taxane or taxane derivative together with a ⁇ -Syn expression inhibitor, preferably a TCA or derivative thereof such as DMI.
  • the combination treatment can include administering an effective amount of a ⁇ -Syn expression inhibitor, preferably a TCA or derivative thereof, such as DMI prior to administration of an effective amount of the chemotherapeutic agent, such that ⁇ -Syn expression is inhibited at the time that the chemotherapeutic agent is administered.
  • a ⁇ -Syn expression inhibitor preferably a TCA or derivative thereof, such as DMI
  • the administration of—Syn expression inhibitor and chemotherapeutic agents can be repeated in alternating or overlapping cycles to achieve a further effect.
  • TCA's such as desipramine
  • chemotherapeutic agents when used in combination with a TCA such as DMI. Accordingly, the methods of treatment described above can also permit using lower than the FDA recommended dosages of the chemotherapeutic agent.
  • a method for identifying improved methods of killing cancer cells that express ⁇ -Syn can comprise determining the ability of an agent to inhibit ⁇ -Syn expression in ⁇ -Syn expressing cancer cells and determining the ability of the ⁇ -Syn expression inhibiting agent to enhance the cytotoxic effectiveness of a chemotherapeutic agent, for example a MT targeting chemotherapeutic agent, and/or a mitosis checkpoint targeting agent, for example a taxane or taxane derivative, in killing the ⁇ -Syn expressing cancer cells.
  • a chemotherapeutic agent for example a MT targeting chemotherapeutic agent
  • a mitosis checkpoint targeting agent for example a taxane or taxane derivative
  • An improved method of killing cancer cells can comprise co-administration of an agent effective to inhibit ⁇ -Syn expression identified by such a method with a chemotherapeutic agent whose effectiveness is increased by inhibition of ⁇ -Syn, or pretreatment with the agent effective to inhibit ⁇ -Syn expression followed by administration or co-administration of the chemotherapeutic agent such that ⁇ -Syn expression is inhibited at the time that the chemotherapeutic agent is administered.
  • Cancer cell lines were tested to assess if inhibitors of ⁇ -Syn expression could be an effective treatment in reducing ⁇ -Syn levels in cancer cells, and if reducing ⁇ -Syn expression also resulted in diminishing their viability.
  • FIG. 4 Two breast carcinoma cell lines were tested for ⁇ -Syn expression by Western blots, and human brain (hIFG) was used as a positive control.
  • T47D cells expressed high levels of ⁇ -Syn ( FIG. 6 ), while SK-BR-3 cells had low ⁇ -Syn levels.
  • Pretreatment of T47D cells for 48 h with 10 or 50 ⁇ M DMI resulted in a decrease of ⁇ -Syn levels, by 40 and 85%, respectively.
  • FIG. 6 These results show that DMI can reduce the protein levels of ⁇ -Syn in endogenously expressing BC cells. Moreover, such reduction occurred in the absence of the norepinephrine transporter, suggesting that DMI has a direct effect on ⁇ -Syn independent of the transporter.
  • DMI+taxol reduces T47D cell viability.
  • FIG. 7 T47D cells were treated with varying levels of taxol or DMI for 48 h and cell viability was measured through MTT assays. In the presence of taxol alone, cell viability was high (52% at 25 ⁇ M taxol), consistent with resistance of ⁇ -Syn-expressing cells to taxol. Cells treated with DMI alone also were similarly resistant to DMI (58% cell viability at 25 ⁇ M DMI). However, at 50 ⁇ M DMI, cell viability was only ⁇ 5% (data not shown) suggesting that DMI alone at high concentrations can be useful in inducing cell death. DMI+taxol reduces SK-BR-3 cell viability.
  • FIG. 8 T47D cells were treated with varying levels of taxol or DMI for 48 h and cell viability was measured through MTT assays. In the presence of taxol alone, cell viability was high (52% at 25 ⁇ M taxol), consistent with resistance of ⁇ -
  • Cells were selected that expressed ⁇ -Syn.
  • the selected cells were treated with varying concentrations of DMI (0-50 ⁇ M) for 48 h.
  • the treatments resulted in a dose-dependent decrease in ⁇ -Syn protein expression; at 50 ⁇ M DMI, >85% of ⁇ -Syn protein expression was decreased.
  • IC50 values for treatment with DMI alone and in combination with taxol demonstrate the synergistic effect in ⁇ -Syn expressing cell lines.
  • FIG. 4 Cell viability assays were conducted as described above for a variety of cancer cell lines that express ⁇ -Syn. ( FIG. 4 ). Treatment with desipramine and taxol showed a synergistic effect of the combined treatments. FIGS. 6-11 . Imipramine also demonstrated a synergistic effect in combination with taxol on cytotoxicity of T47D breast cancer cells.
  • FIG. 13 Various pancreatic cancer cell lines were assayed for ⁇ -Syn expression.
  • FIGS. 14-16 Various pancreatic cancer cell lines were assayed for ⁇ -Syn expression.

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